Abstract: Project 3 addresses two approaches to inducing human immune tolerance to the pig, namely mixed xenogeneic chimerism and porcine thymic transplantation, in a humanized (HU) mouse model. We have demonstrated that each approach can centrally tolerize human T cells to the donor pig and we hypothesize that the combination of both approaches will ultimately be optimal. During the current funding period, we have obtained evidence that mixed xenogeneic chimerism leads to specific tolerance or global unresponsiveness of human NK cells. We have obtained evidence that mixed xenogeneic chimerism tolerizes human T cell- independent B cells producing anti-pig xenoantibodies. We have shown that human T cells developing in human and porcine thymus grafts are functional and dependent on human APCs in the periphery for homeostasis. Human T cells developing in porcine thymus grafts, while tolerant of the donor pig, have reduced homeostatic expansion and reduced responses to antigen presented by HLA. We hypothesize that such abnormalities may be corrected while optimal protective immunity and tolerance to the pig and the human will be achieved by injecting autologous thymic epithelial cells (TECs) into the porcine thymic graft and combining transplantation of this ?hybrid? thymus with mixed xenogeneic chimerism. We will: 1) Engineer a pig/human ?hybrid? thymus to mediate positive and negative selection of human T cells on both pig and human MHC molecules. We will evaluate the impact of a pig/human TEC-containing hybrid thymus and of mixed xenogeneic chimerism on tolerance, homeostasis and function of human T cells. These studies will be directly relevant to the baboon studies in Project 1 and are expected to achieve optimal tolerance and protective immunity; and 2) Determine the impact of duration of mixed xenogeneic chimerism on tolerance of human T, B and NK cells to the pig. We will determine the extent to which human CD47 expression on pig bone marrow prevents pig bone marrow rejection by human macrophages and enhances porcine chimerism in HU mice. We will then separately examine the impact of the duration of chimerism on the persistence of T cell, B cell and NK cell tolerance. We will examine the mechanism of persistent T cell tolerance if observed after loss of chimerism. We will also determine the impact of duration of mixed xenogeneic chimerism on human NK cell tolerance and of transgenic expression of HLA-E by pig bone marrow cells on induction of mixed chimerism in HU mice and on tolerance and function of human NK cells. Collectively, the studies will continue to guide the tolerance studies in Projects 1 and 2 and the development of optimal transgenic pigs in Project 4.